U.S. patent number 5,985,555 [Application Number 08/704,682] was granted by the patent office on 1999-11-16 for method and apparatus for processing nucleic acids using a small temperature-changing zone.
This patent grant is currently assigned to Boehringer Mannheim GmbH. Invention is credited to Wolf Bertling.
United States Patent |
5,985,555 |
Bertling |
November 16, 1999 |
Method and apparatus for processing nucleic acids using a small
temperature-changing zone
Abstract
Method and device for processing nucleic acids in a reaction
mixture on a surface that can be temperature-controlled and its
immediate vicinity wherein the main space of the reaction mixture
remains essentially isothermal. The method has the advantage of a
very short processing time.
Inventors: |
Bertling; Wolf (Erlangen,
DE) |
Assignee: |
Boehringer Mannheim GmbH
(Mannheim, DE)
|
Family
ID: |
6513253 |
Appl.
No.: |
08/704,682 |
Filed: |
September 19, 1996 |
PCT
Filed: |
March 16, 1995 |
PCT No.: |
PCT/EP95/00975 |
371
Date: |
September 19, 1996 |
102(e)
Date: |
September 19, 1996 |
PCT
Pub. No.: |
WO95/25592 |
PCT
Pub. Date: |
September 28, 1995 |
Foreign Application Priority Data
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|
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|
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Mar 19, 1994 [DE] |
|
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44 09 436 |
|
Current U.S.
Class: |
435/6.18;
422/68.1; 435/287.2; 435/6.1; 435/91.1; 435/91.2 |
Current CPC
Class: |
B01L
7/52 (20130101) |
Current International
Class: |
B01L
7/00 (20060101); C12Q 001/68 (); C12P 019/34 ();
C12M 001/34 () |
Field of
Search: |
;435/6,91,287.2,91.2,316,91.5,810,289,290,299.1 ;536/24.3,24.33
;935/6,77,78,87,88 ;165/267,268 ;436/50 ;422/68.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO93/13220 |
|
Jul 1993 |
|
WO |
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WO93/25912 |
|
Dec 1993 |
|
WO |
|
Primary Examiner: Horlick; Kenneth R.
Attorney, Agent or Firm: Nikaido Marmelstein Murray &
Oram, LLP.
Claims
I claim:
1. A method for rapid processing of nucleic acids in a reaction
mixture, the method comprising
temperature-regulating the reaction mixture by changing a
temperature of a temperature-changing element to effect a
temperature change in a temperature-changing zone which adjoins the
temperature-changing element, while maintaining the remaining
portion of the reaction mixture at essentially isothermal
conditions throughout the temperature-regulating step, wherein the
temperature-changing element has a surface which adjoins the
temperature-changing zone, the temperature-changing zone is
relatively small as compared to the entire reaction mixture and
comprises an area less than 0.2 mm in length from the surface, and
reagents capable of binding to the nucleic acids to be processed
are bound to the surface.
2. The method of claim 1, wherein the reagents are
oligonucleotides.
3. A method for detecting nucleic acids in a reaction mixture,
comprising
providing a reaction mixture containing nucleic acids to be
detected and reactants for processing the nucleic acids in a
processing procedure;
temperature-regulating the reaction mixture by changing
temperatures of a temperature-changing element to effect
temperature changes in a temperature-changing zone which adjoins
the temperature-changing element, while maintaining the remaining
portion of the reaction mixture at essentially isothermal
conditions throughout the temperature-regulating step, to amplify
any nucleic acids in the temperature-changing zone, wherein the
temperature-changing element has a surface which adjoins the
temperature-changing zone, the temperature-changing zone is
relatively small as compared to the entire reaction mixture and
comprises an area less than 0.2 mm in length from the surface, and
reagents capable of binding to the nucleic acids to be processed
are bound to the surface; and
detecting the amplified nucleic acids.
4. The method of claim 1, wherein at least one reaction step
involved in the processing of the nucleic acids proceeds at an
increased temperature in the temperature-changing zone.
5. The method of claim 1, wherein the temperature change in the
temperature-changing zone is at least partially accomplished by
convection or by stirring the reaction mixture.
6. The method of claim 1, wherein the reaction mixture further
comprises further reagents capable of binding to the nucleic acids
to be processed.
7. The method of claim 1, in which at least one processing step
requires a temperature that differs from the temperature of the
reaction mixture.
8. The method of claim 7, wherein a temperature-changing step
serves for thermal denaturation.
9. The method of claim 7, wherein a temperature-changing step
serves to hybridize nucleic acids.
10. The method of claim 7, wherein a temperature-changing step
serves to extend a template molecule.
11. The method of claim 7, wherein various processing steps serve
to target sequence-dependently amplify a template molecule.
12. The method of claim 11, wherein a non temperature-resistant DNA
polymerase is used for the amplification.
13. The method of claim 11, wherein a temperature-resistant DNA
polymerase is used for the amplification.
14. The method of claim 11, wherein RNA-dependent RNA polymerase is
used for the amplification.
15. The method of claim 11, wherein RNA-dependent DNA polymerase is
used for the amplification.
16. The method of claim 11, wherein DNA ligase is used for the
amplification.
17. The method of claim 1, wherein the volume of the reaction
mixture is between 20 .mu.l and 1 ml.
18. A system for processing nucleic acids in a reaction mixture,
comprising
a reaction mixture containing nucleic acids to be detected and
reactants for processing the nucleic acids in a processing
procedure, and
a temperature-changing element which effects temperature changes in
a temperature-changing zone of the reaction mixture while
maintaining the remaining portion of the reaction mixture at
essentially isothermal conditions, wherein the temperature-changing
element has a surface which adjoins the temperature-changing zone,
the temperature-changing zone is relatively small as compared to
the entire reaction mixture and comprises an area less than 0.2 mm
in length from the surface, and binding means for binding agents
for processing the nucleic acids are bound to the surface.
19. The system of claim 18, wherein the binding means is a chemical
or physical binding reagent.
20. The system of claim 18, wherein the binding means is a nucleic
acid binding reagent and the agents for processing the nucleic
acids are nucleic acids that are complementary to at least a part
of the nucleic acids to be processed.
21. The system of claim 18, wherein the temperature-changing
element is time-coordinated to effect time-coordinated temperature
changes in the temperature-changing zone.
22. The system of claim 18, wherein the binding of the agents is
achieved by chemical or biospecific binding.
23. The system of claim 18, wherein the binding means is magnetic
particles.
24. The system of claim 23, further comprising a magnet, located
behind the surface, which induces a magnetic field to attract the
magnetic particles.
25. The system of claim 18, wherein the temperature-changing
element is a single-use device.
26. The system of claim 18, wherein only the surface of the
temperature-changing element is single-use, and the remainder of
the temperature-changing element can be used several times.
27. The system of claim 18, wherein the surface protrudes into the
reaction mixture.
28. The system of claim 27, wherein the surface can be cooled
and/or heated.
29. The system of claim 18, wherein the temperature of the surface
is regulated by a control unit.
30. The system of claim 18, wherein the temperature-changing
element comprises a heating element with a small heat capacity and
a cooling element with a high heat capacity.
31. The system of claim 30, wherein the heating element is heated
ohmically.
32. The system of claim 30, wherein the heating element is heated
by rays.
33. The system of claim 32, wherein the rays are infrared rays.
34. The system of claim 32, wherein the rays are laser-generated
rays.
35. The system of claim 18, wherein the surface is composed of a
foil made of a heat-conducting material.
36. The system of claim 35, wherein the heat-conducting material is
metal.
37. The system of claim 36, wherein the heat-conducting material is
gold.
Description
The invention concerns methods for processing nucleic acids by
means of a temperature regulation element as well as devices and
instruments for carrying out these methods.
In analytics and especially in medical diagnostics more and more
methods are beginning to be established which are based on nucleic
acid tests and syntheses. Nucleic acids are for example very well
suited as a very specific detection agent for organisms for the
diagnosis of diseases. During these detection methods various
processing steps such as denaturation, hybridization, syntheses and
immobilization of nucleic acids and enzymatic treatment thereof are
common. For a long time a problem of such methods was the small
amount of nucleic acids in the samples.
A solution for this problem, which has made it possible to detect
numerous analytes, was the amplification of nucleic acids. Such a
method is described in EP-A-0 200 362 i.e. the polymerase chain
reaction (PCR). In this method many copies of the original nucleic
acid are produced by repeated extension of primers in a reaction
solution. These can for example be detected by hybridization with a
labelled nucleic acid probe according to EP-A-0 201 184. The
reaction solutions used for this are heated to particular
temperatures and cooled again at intervals in order to separate
double strands and for the extension reaction. Since the volumes
are relatively large, the times required for temperature regulation
result in a relatively long period to carry out the entire
amplification process.
It has been attempted to remedy this by so-called capillary PCR. In
this method the reaction mixture is present in glass capillaries of
a small diameter. As a result the time required to carry out an
amplification sequence can be reduced to ca. 30 min. A problem with
capillary PCR is the delicacy of the glass of the capillary which
also has to be heated and the cumbersome sample application.
Recently more and more amplification processes are also being
described in which the amplification reactions can be carried out
in a closed system i.e. without supplying reagents during the
cycles. Thus in a system is described WO 92/07089 in which the
amplification mixture is passed as long as necessary through a
circulation system in which the reaction mixture is repeatedly
heated and cooled. The period in which the mixture is retained in
the individual zones of the system can be influenced by selecting
the diameter. An amplification process is described in EP 0511712A1
in which the amplification mixture is brought cyclically to quite
specific temperatures in order to achieve relatively short cycle
times. In this case also the sample handling is made more
difficult.
The object of the present invention was among others to improve the
existing nucleic acid processing methods and in particular to
provide processes in which the amplification can be completed in a
particularly short time.
The invention therefore concerns a method for processing and in
particular amplifying nucleic acids in a reaction mixture
characterized in that the temperature of a surface adjoining the
reaction mixture and its immediate surroundings is regulated but
the main space of the reaction mixture remains essentially
isothermal.
The invention also concerns a device for processing and amplifying
nucleic acids with a temperature regulation element as well as an
instrument which contains this device.
Nucleic acids within the sense of the invention are all types of
nucleic acids, modified or unmodified. Unmodified nucleic acids are
for example the naturally occurring nucleic acids. Modified nucleic
acids can be formed by substituting groups of the natural nucleic
acids by other chemical residues. Examples are nucleic acid
phosphonates or phosphothioates and nucleic acids modified on their
sugar residues or bases by chemical groups which may also be
detectable.
The processing of nucleic acids according to the present invention
preferably includes at least one reaction step which proceeds at an
increased temperature that is at least different from the ambient
temperature. Such reaction steps are for example the thermal
denaturation of partially or completely double-stranded nucleic
acids. In this process the nucleic acids are heated to temperatures
above the relevant melting point in order to produce single strands
or to melt secondary structures. A further processing step of
nucleic acids is the hybridization of the single-stranded regions
of nucleic acids which are complementary to one another to form a
nucleic acid double strand (hybrid). This takes place at
temperatures below the melting point of the hybrid. Therefore
in-order achieve a hybridization it is often necessary to cool the
reaction mixture. Especially in amplification or sequencing methods
a further processing step is carried out namely the extension of a
primer hybridizing to a so-called template nucleic acid with the
aid of further mononucleotides or oligonucleotides. In t his case
the temperature is preferably adjusted to that at which the enzyme
used has its activity optimum or at which competing reactions are
reduced. This temperature may also be identical to the
hybridization temperature (2-step PCR).
A special case for processing nucleic acids is the amplification of
nucleic acids. In this connection practically all amplification
methods e.g. target sequence-dependent amplifications (in
particular non-isothermal amplifications such as the polymerase
chain reaction, ligase chain reaction or similar ones) can be
carried out according to the invention. Also during these
amplification methods at least one of the aforementioned
temperature sensitive processing steps takes place.
A key feature of the present invention is the fact that the change
of temperature does not take place in the entire reaction mixture
but only in a very small part of the reaction mixture. As a result
the heating and cooling of this relatively small zone can occur
very rapidly thus considerably accelerating the processing of
nucleic acids. Therefore in order to carry out the process
according to the invention the reaction mixture is contacted with a
surface whose temperature can be regulated. By changing the
temperature of the surface the immediate surroundings of this
surface are heated, equalized or cooled depending on the processing
step and temperature of the reaction mixture. One can differentiate
between several cases.
If the temperature of the reaction mixture is higher than the
melting temperature of the nucleic acids to be processed, the
surface can be cooled in order to achieve a hybridization of the
nucleic acids in the adjoining vicinity. If the temperature of the
reaction mixture is lower than the melting temperature and the
temperature which is required to extend a primer on the nucleic
acid, the surface and thus the immediate vicinity can be firstly
heated to a temperature at which an extension of the primer can
take place. Subsequently the temperature can be increased to above
the melting point of the nucleic acids which enables a denaturation
and strand separation of the double-stranded nucleic acids which
have been formed. This can be followed by cooling to a temperature
at which the single strands can hybridize with new primers. This
cycle can be repeated several times.
If the temperature for the reaction mixture lies between the
optimal temperature for extension and the melting temperature, the
temperature at the surface is firstly increased in order to
separate double strands. Subsequently the temperature is lowered to
a temperature at which the single strands hybridize with primers.
Subsequently the temperature is adjusted to an optimum for the
extension and if desired the cycle is repeated.
The desired temperature can be maintained over a predetermined
period e.g. until the desired reactions have taken place at the
surface. For this purpose the temperature of the surface can also
be kept constant by known control measures and connected regulation
measures (reheating, recooling). The time periods depend, as is
usual for the known processes, on the length of the nucleic acids
to be processed and their homology as well as on the special
hybridization conditions. However, a person skilled in the art can
determine the optimum periods by simple experiments. The time
periods are between fractions of seconds and a few seconds
depending on the processing step.
In the above mentioned cases the main space of the reaction mixture
remains essentially isothermal whereas the reaction mixture located
in the immediate vicinity of the surface adjusts to the set
temperatures of the surface.
In order to regulate the temperature and to set specific
temperatures at the surface and its immediate vicinity it is
recommended that a surface of a device is selected as the surface
that is composed of a heating element and a cooling element. The
heating element preferably has a relatively large surface with a
comparatively small heat capacity. Metal foils have for example
proven to be suitable which can be heated in a suitable manner e.g.
by electrical current. Good heat conducting materials such as gold
are preferred as materials for the metal foil.
The cooling element should have a relatively high heat capacity.
Solid, liquid or gaseous substances come into consideration as the
cooling medium. Liquid cooling media such as water are preferred. A
good heat conductivity is advantageous.
The arrangement of the heating element and cooling element can be
changed according to the temperature of the reaction medium
depending on whether it is intended to use the device more for
heating or for cooling the surface and its immediate vicinity.
However, in general the heating element is located in the immediate
vicinity of the surface. The surface of the heating element can
also directly adjoin the reaction mixture. However, it is also
possible to separate the heating element from the reaction mixture
by a thin heat conducting layer.
The cooling element can in principle be positioned at any position
which allows a cooling of the surface and the immediate vicinity.
However, it has proven to be advantageous to position the cooling
element on the opposite side of the reaction mixture to the heating
element. If the heating element has a low heat capacity, the fact
that the heating element must also be cooled is not an important
disadvantage.
In the method according to the invention the heating element is
used to heat until the surface and its immediate vicinity have been
heated to the desired temperature. In this process a pronounced
temperature gradient will form near the surface whereas the
remaining part of the reaction mixture remains isothermal. This
applies in particular if there is no significant exchange of liquid
during the heating process but even if there is an exchange of
liquid. The heating process can be completed within fractions of a
second and in favourable cases even is milliseconds. The same
applies to the cooling. The reaction space which is heated to the
desired temperature can be very small, it is preferably less than
0.2 mm particularly preferably less than 0.5 .mu.m deep. The area
of the heating element facing the reaction mixture influences the
depth of the temperature gradient and its rate of formation. A
preferred size of the surface may result from the desired
application of the process. Usually the surface is <2 cm.sup.2,
particularly preferably between 0.2 cm.sup.2 and 0.2 mm.sup.2. The
surface can be smooth or also rough. If the surface is also used at
the same time as a carrier for agents for processing nucleic acids
it is advantageous to enlarge this surface by surface
structures.
The volume of the reaction mixture is of no major practical
importance for the invention. It may be a drop with a volume of 20
.mu.l but also a volume of any size. An advantage of the process
according to the invention is that the device containing the
heating and cooling elements can for example also be inserted into
a large vessel containing the reaction mixture in which case the
key reactions only take place in a very small boundary region of
the surface whereas the remaining reaction space exerts almost no
influence on the reaction. On the other hand the samples that are
available and amounts of reagent represent a practical limit to the
size of the reaction mixture. The reaction volumes will therefore
usually lie in a range between 1 ml and 30 .mu.l preferably between
100 .mu.l and 50 .mu.l but it is also for example possible to use
very much smaller volumes by applying the reaction mixture onto the
temperature-regulating surface.
In order to process nucleic acids these are applied to the
temperature-controllable surface or its immediate vicinity. This
can be achieved in various ways for example mechanically or by
diffusion. The nucleic acids are preferably bound to the surface.
This binding can in turn be of various types. A chemical binding is
preferred either via adsorption or biospecific interactions. In the
case of binding by adsorption, the surface can be covered with a
nucleic acid binding reagent. Biospecific interactions can be
interactions between nucleic acids and the nucleic acids to be
processed (hybridization, complementary part of these nucleic
acids) but also interactions between antigens or haptens and
antibodies directed against them and receptor-ligand interactions.
A preferred type of binding of the nucleic acids to be processed to
the surface is via oligonucleotides which are bound covalently to
the surface and which are complementary to at least part of the
nucleic acid to be processed. However, these oligonucleotides can
also be bound to the surface by means of biospecific interactions
e.g. biotin-streptavidin.
According to the present invention the binding of the nucleic acids
to be processed is preferably reversible. The nucleic acids can be
released again after a period depending on the process which is to
be carried out by heating the surface and its immediate vicinity.
Any desired step can be carried out between the binding and release
of the nucleic acid such as chemical reactions and also a
separation of the bound nucleic acids from the original reaction
mixture and transfer into a new reaction mixture.
In the case of an amplification reaction of nucleic acids the
oligonucleotides bound to the surface can be used as primers for
elongation or extension using the nucleic acid to be processed as a
template. As a result a bound extended primer forms on the nucleic
acid to be processed. The nucleic acid used as a template can be
detached from the extension product by increasing the temperature
and in the next temperature cycle it can act as a template for a
new immobilized primer which has not yet been elongated. In this
manner it is possible to extend a large amount of primers
immobilized on the surface and thus produce copies of parts of the
nucleic acid to be processed. The extended (elongated) primers
attached to the surface in turn serve to multiply the molecule
serving as a template by means of complementary primers. The
reagents required to carry out the reactions have either to be kept
on hand in the total reaction mixture or they must be added as
required. Therefore for an amplification reaction according to the
principle of a polymerase reaction (EP-B-0 201 184) the
deoxyribonucleotides, a DNA polymerase and a further primer and
suitable buffer reagents must be kept in the reaction mixture. In
order to prepare other nucleic acid types (e.g. RNA) other reagents
e.g. ribonucleotides or RNA-dependent polymerases are provided.
This step of the process can take the working temperature of the
processing enzyme into particular consideration.
The nucleic acids to be processed can, however, also be bound to
the surface by physical methods e.g. by means of a magnet. For this
the nucleic acids must, however, be bound to a magnetizable
particle. The binding of magnetic particles coated with nucleic
acid can be carried out in a reversible manner by locating or
inducing a magnet behind the surface. By applying an alternating
field it is possible to bind the magnetic particles to the surface
or remove them therefrom.
Special embodiments which have some advantageous effects are also
conceivable. Thus the efficiency can be increased by increasing the
diffusion of the nucleic acids in the reaction mixture by
convection. It may also be advantageous to use higher
concentrations of the reactants compared to the reaction mixtures
usually used. In addition the nucleic acid to be processed can be
concentrated at the surface at the start of the reaction by
prehybridization.
In the case of an amplification or multiplication of nucleic acids
it may be that the number of cycles to be carried out e.g. in a PCR
has to be increased in order to produce sufficient amplification
product. However, due to the very short cycle time according to the
process of the invention this is not a disadvantage.
If the reaction mixture is kept at a relatively low temperature
this means that less demands have to be made on the heat stability
of the reagents than when the entire reaction mixture is heated
several times. Therefore a thermostable polymerase is not
absolutely necessary for an amplification reaction and nevertheless
it is not necessary to pipette new enzyme into the reaction mixture
in each amplification cycle.
Any desired further steps can follow the processing of nucleic
acids according to the invention. Thus the nucleic acids can be
examined either in a bound state on the surface or in a released
state or processed with further reagents.
Thus with the aid of the described amplification process according
to the invention it is possible in a simple manner to develop a
process for detecting nucleic acids in a sample. For this the
surface to which the oligonucleotides acting as a primer are bound
is contacted with the sample liquid. Afterwards the temperature of
the surface and its immediate vicinity is brought to a temperature
which lies above the melting point of the double-stranded nucleic
acid contained in the sample. After cooling the surface and its
immediate vicinity the nucleic acid to be detected is hybridized
with the oligonucleotide. By means of the cycle process as
described above numerous copies are produced with the aid of the
nucleic acid to be detected which can remain bound to the surface
at the end of the amplification reaction. The amount of nucleic
acids on the surface can be determined by hybridization with
labelled nucleic acid probes and detecting them with the aid of the
label. In the case of methods which allow a direct detection of
nucleic acid hybrids without a label or of extended single strands
(primers) (e.g. according to the principle of surface plasmon
resonance) a direct detection is also possible. If labelled primers
or mononucleotides are used for the amplification reaction
hybridization with a detectable labelled nucleic acid probe is
omitted. An approach is also conceivable in which a higher
temperature is maintained in the mixture and the surface adopts the
lower temperatures necessary for the further steps.
Since the application of the process according to the invention to
amplify nucleic acids proceeds practically on a surface, we shall
introduce the name 2D amplification for this amplification
reaction.
The invention also concerns a device which can be used in the
aforementioned processing method. In particular the invention
concerns a device for processing nucleic acids by means of a
temperature regulation element, this element being suitable for
regulating the temperature of the surface and its immediately
adjoining vicinity and wherein agents can be bound to this for
processing the nucleic acids. These agents can be oligonucleotides
and serve for example as primers. This device preferably contains a
cooling element as well as a heating element in which the
arrangement is preferably as in the processing method described
above. This device is preferably very small. It can for example
have a thickness of <5 mm and an area of <10 cm.sup.2. The
elements located therein such as the cooling element or the heating
element are simple components. Therefore this device is excellently
suitable for single use (disposable) which can reduce the risk of
contamination that is inherent to re-usable devices. If the device
is to be suitable for multiple use, it is also possible to separate
the heating element by a very thin component from the space which
contains the reaction mixture and to make this component separable
from the device. A new component can then be inserted for a further
processing step.
The invention also concerns an instrument for processing nucleic
acids which contains a control element for a time-dependent
temperature regulation as well as a device according to the
invention. The control element of a so-called thermocycler
according to EP-A-0 236 069 can be used as the control element;
however, a control according to the principle of ink-jet printers
is preferred since they have a higher control rate. The control
element must be able to heat the heating element at predetermined
intervals until the vicinity of the surface has an adequate
temperature. It must also be able to provide a cooling of the
surface by means of a cooling element at predetermined intervals.
For this a cooling liquid can for example be passed through the
device. Provided the heat capacity of the heating element is small
but the heating efficiency is relatively high, a continuous cooling
is also possible. Various forms can be used as the surface in an
ohmically temperature controlled version of this instrument which
can be thermally heated depending on the voltage and the duration
of the current. In a modified version the surface to be temperature
controlled is located on a preferably black support, preferably a
plastic foil, which is heated by a laser ray, preferably an
infrared laser, on the side distal to the surface to be temperature
controlled. The surface to be temperature controlled as well as the
heat-absorbent and heat-conducting material can in this case be
mounted on a support surface preferably glass that is permeable to
infrared.
The device according to the invention can have various embodiments.
For example it can be designed to be immersed in a vessel. However,
it can also be designed such that the liquid containing the nucleic
acid to be processed is dripped onto the surface or is dispensed
into a vessel formed by the surface. This reaction space can also
be closed after filling it with the liquid so that contamination
problems can be reduced.
One embodiment of the invention is a process for amplifying nucleic
acids. This is as in example 3, for example an amplification
reaction denoted polymerase chain reaction with subsequent
detection.
A further embodiment of the process according to the invention is a
process for concentrating nucleic acids e.g. by hybridization. When
primers with an adequate specificity are adsorbed covalently onto
the surface, it is possible to preset a hybridization temperature
when the reaction solution to be analysed has a known composition
(ionic composition, concentration of reagents) such that the
nucleic acid to be analysed binds specifically to the primers bound
covalently to the surface for hybridization. This process can be
used
a) to exactly analyse the actual hybridization temperature
b) to achieve an exact concentration of the nucleic acid to be
analysed in the reaction mixture
c) to test for the presence of cross-hybridizing sequences.
This short list is not definitive.
This process can also be used to transfer nucleic acids from one
solution into another. Then a hybridization takes place in the
first vessel (lower temperature) and a denaturation in the second
vessel (higher temperature).
A further embodiment of the present invention is a process for
sequencing nucleic acids. One method of applying the invention in
relation to sequence analyses is the so-called minisequence
analysis. In this method the exact sequence of the nucleic acid
nucleotide adjoining the primer can be determined by adding
exclusively dideoxynucleotides and no deoxynucleotides for
sequencing to the solution which is provided for sequencing. The
four possible dideoxynucleotides ddATP, ddCTP, ddGTP and ddTTP are
labelled with different fluorescent labels. In each case the
incorporation of the next respective nucleotide thus leads to a
termination of the sequence reaction and, after purification of the
extended primer located at the surface, the sequence of the
nucleotide located on the primer can be determined by analysis of
the specific fluorescence.
A special case for the possible application of the present
invention is a process for sequencing nucleic acids which have
previously been amplified in a PCR reaction. After the
amplification the amplified product is present covalently bound via
the primer to the reactive surface in double-stranded form.
Denaturation of this amplificate by a brief passage through a high
temperature phase will make it single-stranded, will allow it to be
purified by transfer to a washing solution and can afterwards be
used again for a subsequent sequencing by transfer this time into a
sequencing reaction. This sequencing is particularly efficient
since now only single-stranded template material is present to
which a sequencing pair binds particularly efficiently. As a result
of the sequencing reaction a double-stranded molecule is
subsequently again present whose labelled (sequenced) half is now
available for analysis by gel chromatography. By using different
labels (with dideoxynucleotides labelled with different fluorescent
markers) all four reactions necessary for a sequence analysis can
be carried out simultaneously; with the conventional method this
analysis has to be repeated four times.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is elucidated in more detail by the following
figures:
FIG. 1 shows the construction diagram of a device according to the
invention (1) which dips into a reaction vessel (3) filled with the
reaction mixture (2). The device contains a cooling element (4) and
a heating element (5) heated by means of a current.
Oligonucleotides (6) are covalently bound to the surface of the
heating element. The cooling element as well as the heating element
can be regulated via connections (7; 8) to control units.
An example of a temperature curve for an amplification reaction of
the PCR type is shown in FIG. 2. In a first phase (A) the nucleic
acids are denatured at a temperature of slightly below 100.degree.
C. In a phase (B) the nucleic acids to be amplified are hybridized
with the immobilized oligonucleotides on the solid phase at about
50.degree. C. In a third phase (C) the primers are extended at ca.
70.degree. C. using the nucleic acid as a template.
FIGS. 3A-3C shows the reactions occurring in the immediate vicinity
of the surface (phases A to C). Three isotherms are shown as a
schematic time course for phase (A) and only one isotherm is shown
for phase (B) and (C) in each case. The nucleic acids are denatured
in phase (A) and diffuse for a certain time as single strands, in
phase (B) templates and primers hybridize and in phase (C) primers
are extended along the templates.
FIG. 4 shows a possible prototype of a surface temperature control
suitable for an application according to the invention.
The heating unit which contains the replaceable heated gold
surfaces as well as the physical requirements for cooling or
heating (connection for cooling agent, electrical connection) is
attached via plug connections (7; 8) to the actual measuring unit
(1) to which holders for reaction vessels (11) are also attached in
which the template molecules, buffer components and primers in
solution and the polymerase required for carrying out the reaction
are present.
The measuring sensors (9) in this case in the form of a test strip
contain as an example gold surfaces (5) in FIG. 4 which do not have
to be coated with the same primers (6).
FIG. 5 shows enlargements of the measuring units with replaceable
test strips from FIG. 4 in which case the measuring unit (1,
composed of 9 and 10) is composed of an electronic connection and
cooling system (upper, grey part of the figure) and the test strip
(=measuring casing). The lower part of the figure shows a
cross-section through this measuring unit which shows the cooling
circulation (4) used for the cooling with a stirring mechanism (12)
and cooling liquid (13) as well as the reaction surfaces (5, 6)
with an electronic connection. The right hand part of the lower
figure shows a further enlargement of the cathode connection of the
gold membrane (5) with a supporting and separating filter (14) as
well as an adhesion layer for the oligonucleotides (6).
FIG. 6 is composed of a diagram of the temperature gradients formed
in the immediate vicinity of the adhesion layer containing
oligonucleotides and the temperature profile which is to be
expected, in this case as exemplified by a three-step PCR in which
temperatures of 96.degree. C. for 0.1 seconds, 54.degree. C. for
0.5 seconds and 72.degree. C. again for 0.5 seconds are shown.
The following examples are intended to further elucidate the
invention in more detail:
EXAMPLE 1
Manufacture of a Surface that can be Cyclically
Temperature-Controlled (Variant a)
An exemplary gold surface that can be cyclically
temperature-controlled can be obtained by milling out two parallel
oblong holes of ca. 1 mm width and 3 mm length at a distance of 3
mm to one another in a thin printed-circuit board. On the side
which is facing the solution and which is therefore called the
proximal side these oblong holes are connected to one another by
vapour-deposited gold. On the distal side one of these oblong holes
is connected to the anode via the circuit-board conductors the
other is likewise attached to the cathode via the circuit-board
conductors.
The gold layer on the proximal side is applied by applying a mask
of the desired size, in this case 3.times.3 mm flush, over the two
longitudinal surfaces. The inner sides of the longitudinal surface
are galvanically coated with copper flush up to the surface. In the
following vapourization process which is carried out conventionally
the two electrically conductive longitudinal holes are now coated
with only a few .mu.m thick gold layer. The coating process is
described in the following:
The gold surface is manufactured by vapour-depositing on "Lexan"
polycarbonate foils (manufacturer: General Electric, thickness 0.75
mm) with dimensions of 8.times.8 cm over a metal mask (d=0.5 mm,
aluminium) in a Leybold high-vacuum coating apparatus (Univex 450).
The surfaces lie at regular distances which enables the
vapour-coated plate to be separated into several units. The
thickness of the gold layer is 300 nm. In a further step the
multi-gold spot plate is coated with "dilute" biotinthiol binding
layers to form a hydrophobic SAM layer. The biotinthiol compound
(HS-C12-DADOO-biotin;
N,N'-(12-mercaptododecylyl-biotinylyl)-2,2'-diaminoethylglycol
diether; 2.94 mg; 5.times.1.sup.0-5 m) and the diluent
(12-mercaptoundecanol, 9.2 mg, 4.5.times.10.sup.-4 m) were
dissolved in 100 ml ethanol p.a. The freshly coated polycarbonate
foils were immersed in this solution. After 4 hours the plates were
taken out, washed twice with ethanol p.a. and immediately coated
with streptavidin. For this purpose the gold spots and the
SAM-coated foils were immersed for 1 hour in a streptavidin
solution (streptavidin concentration: 0.5 mg/ml in 0.05 M potassium
phosphate buffer pH 7.2). The surfaces treated in this way were
subsequently treated with a washing solution (50 mM potassium
phosphate buffer pH 7.2, 2% sucrose, 0.9% NaCl, 0.3% bovine serum
albumin fraction II) and afterwards dried for 20 h (25.degree. C.
and 40% humidity).
The very small thickness of the gold layer results in a very small
conducting gold cross-section (3 mm.times.3 .mu.m over a length of
3 mm). This gold layer therefore represents a relatively high
resistance compared to the circuit-board conductors.
The conductors are now connected to a current supply which enables
the computer-aided control of the ohmically induced temperature of
the gold surface.
Manufacture of a Surface that can be Cyclically
Temperature-Controlled (Variant B)
In one variant of the manufacture of the surface that can be
cyclically temperature-controlled a gold layer is vapour-deposited
on the covering layer which isolates the measuring meander made of
platinum, a commercially available temperature sensor made of
platinum (PT 100). If this measuring sensor is now connected to a
regulator-controlled voltage source then this sensor can also be
used as a heat source for the vapour-coated gold layer. For this it
is necessary to pass the current and voltage of the platinum sensor
via an analogue divider onto a regulator. The regulated parameter
of the regulator is the resistance of the heated platinum element
which enables an exact temperature control when normalization of
the gold surface temperature with the temperature of the platinum
meander has taken place.
EXAMPLE 2
An Example of a Device to Measure the Surface Temperature of a Gold
Foil (Variant a)
A gold foil according to example 1, variant a is covered with a
mask which allows a ca. 1 mm wide gold thread to be
vapour-deposited on the gold foil which protrudes beyond this gold
surface and can be connected to a measuring point. A further mask
which allows a second thread to be vapour-deposited e.g. of nickel,
bismuth or another alloy suitable for temperature measurement, is
also prepared. The metal thread lies on the same side of the gold
surface as the gold thread (200 nm thick) but its path separates
when leaving the gold surface to be temperature-controlled and it
leads to a second measuring point as a separate metal thread (300
nm thick). This arrangement of the measuring probes made of gold
and a further metal or alloy suitable for temperature measurement
enables the thermovoltage (2.2 mV/100.degree.0 Kalvin) to be
measured in the area of the gold foil where the two threads of the
measuring probe overlap. The temperature at the cold junction is
measured with a thermoprecision PT 1000 element (accuracy >1%)
and standardized to 10 V (0 V corresponds to 0.degree., 10 V
corresponds to 100.degree.), only amplified and also standardized
to 10 V. The sum of the two temperatures is formed in a summation
amplifier and used to electronically regulate the respective
surface temperature.
EXAMPLE 2b
An Example of a Device for Measuring the Surface Temperature of the
Gold Surface From Example 1, Variant b
In order to exactly determine the surface temperature of the gold
layer it is possible to press together the surfaces of two PT 100
measuring sensors vapour-coated with gold as described in example 1
variant b in such a way that half of the gold-coated area of the
heating sensor covers half the area of the measuring sensor and the
remaining area in each case is located in the surrounding medium.
If only one sensor is now used for measuring and the other one for
heating this then allows the temperature of the gold surfaces to be
calculated exactly. The temperature at the surface of the sensors
exactly corresponds to the mean temperature of the heated and
unheated sensor which can both be exactly defined by measuring
their resistance. In this way using a temperature sensor used for
measurement it is possible to calibrate a whole series of
temperature-controllable surfaces and prepare them for carrying out
PCR reactions. The temperature gradient between the heated and
unheated surface of two surfaces arranged in this manner is ca.
3.degree. C.
EXAMPLE 3
Procedure for a Two-Step PCR Using a Primer that is Immobilized to
a Surface
An example of carrying out a polymerase chain reaction uses a
biotin-labelled primer that is immobilized on a surface
(5'-GAAGGGAGGAAGGAGGGAGCGGAC-3'SEQ ID NO: 1). The 5' end of this
primer is coupled via its biotin group to a superficial
streptavidin or gold-streptavidin surface. The molar amount of
streptavidin or biotin/square millimeter is about 0.2
pmol/mm.sup.2. Nanogram amounts or less of the following
double-stranded or single-stranded template molecule (only one
strand is cited
5'-GAAGGGAGGAAGGAGGGAGCGGACGTCCACCACCACCCAACCACCCCACCC-3'SEQ ID NO:
2) and an opposite primer at a concentration of between 0.5 .mu.M
and 2 .mu.M are present in solution. The reaction takes place in
commercial PCR buffer (Boehringer Mannheim, instruction enclosure
for the enzyme, 8th edition, order no. 1146165) at 1.5 mM
Mg.sup.2+. A commercial Taq-DNA polymerase (Boehringer Mannheim,
order no. 1146165) at a concentration of 20 nM is used as the
polymerase. The opposite primer (5'-GGGTGGGGTGGTTGGGTGGTGGTG-3'SEQ
ID NO: 3) present in solution was labelled with digoxigenin at its
5' end (Patent EP 0324474).
The PCR reaction was carried out at a constant solution temperature
of 68.degree. and on a primer-coated surface cyclically varying
between 96 and 68.degree.. The cycle duration of the higher
temperature in our example varied between 0.1 seconds, 10 seconds
and 1 minute, the lower temperature between 0.5 seconds, 20 seconds
and 1 minute.
After completing the cyclic heating the solution was cooled to room
temperature in the process of which the complementary elongated
biotin-labelled and elongated digoxigenin-labelled primer hybridize
to one another and double-stranded DNA fragments are formed. After
washing with PCR buffer solution the surfaces were reacted with
anti-Dig-POD, incubated for 30 minutes, washed again and admixed
with ABTS (colour protocol according to the instruction enclosure
item 3 of the Boehringer Mannheim Reverse-Transcriptase-Assay,
non-radioactive, order no. 1468120). The coloured product that
forms was determined quantitatively in an ELISA photometer at a
wavelength of 405/490 nanometers. The absorbance values obtained
prove an amplification of a region of the template measured as the
extended opposite primer that hybridizes to the primer coupled to
the solid phase. In typical experiments we obtained absorbance
values of 0.4 when measuring in an ELISA reader and after
correcting the digoxigenin-free blank value. The values in control
experiments without template were 0.04 after correcting the blank
value and in controls without polymerase or without temperature
increase but using the complete reaction mixture the values were
0.07. Even when heating the surface for several minutes (5 minutes)
the ambient temperature in the constantly temperature-controlled
solution only increased by 7.degree. C. even when a significantly
(100-fold) larger surface (3 cm.sup.2) was heated.
EXAMPLE 4
Manufacture of a Device According to the Invention
An example of a device according to this invention is composed of
three structural units. These three structural units are
1. the heatable temperature-controllable surface which penetrates
into the liquid reaction medium
2. the components that are necessary for heating and cooling that
are connected to
3. the electronic control.
The surface of the element described in this example is composed of
a thin gold foil with a thickness of less than half a millimeter
which is firmly bound to a streptavidin layer. The biotin-labelled
oligonucleotides required for the further processing steps can
likewise be applied to this dextran layer by means of affinity
coupling. These oligonucleotides are then coupled covalently via
their 5' phosphate end to the surface and thus have a reactive 3'
end which is accessible to enzymes.
This gold foil which can be structurally stabilized by a supporting
plastic net and which has a reactive surface of about 5.times.5 mm
serves at the same time as the heating element since it is
connected to an electric power source which leads to a spontaneous
heating of this gold foil when current flows. The cooling element
is located distally to the reaction mixture, behind the gold foil.
In this case the cooling element is composed of a channel recessed
in plastic which adjoins the gold foil with an area of 7 mm in
width. The channel is 2 mm in depth and runs along the complete
length of that part of the instrument denoted measuring sensor
which in this case comprises the heating element and cooling
element as well as the reactive surface. This channel runs from one
end of the measuring sensor to the other end of the measuring
sensor at which the reactive surface is applied and back again by
inclusion of a cross-piece that separates the two channels. In this
component denoted coolant loop a suitable cooling liquid e.g. water
is circulated. The entire unit is cast in hard plastic and can be
recycled i.e. the gold foil as well as the plastic are
reusable.
The control of the heating the reactive surface as well as of the
circulation rate and precooling temperature of the coolant is
achieved by means of a third electronic component which is attached
outside the measuring sensor. This third unit also comprises the
storage vessel as well as the connectors for the cooling liquid. In
addition there is a micropump in this third unit which is
responsible for circulating the cooling liquid. The measuring
sensor is made ready-to-use by flanging the coolant pipes onto the
measuring unit and connecting the power source to the reactive
surface. The individual temperature ranges as well as the duration
of action influence on this surface can be preselected via an input
unit that is attached to the surface of the control unit and has a
digital display.
EXAMPLE 5
Procedure for an Amplification Reaction with the Aid of the Device
According to the Invention
This example of an application of the device according to the
invention is an amplification reaction of a predetermined nucleic
acid sequence denoted polymerase chain reaction. Two primers each
with a length of 16 bases that code at a distance of 4 nucleotides
to one another have been selected for this nucleic acid sequence.
The sequence of one of the primers is identical to and that of the
other one is complementary to the sequence to be analysed. The
primer denoted identical in this case is coupled to the reactive
surface via a biotin label present at the 5' end. The other
complementary primer as well as all further reagents that are
usually used in a so-called PCR reaction are added to the reaction
mixture. The reaction is started by setting the respective reaction
temperatures that are to predominate in the immediate vicinity of
the reactive surface and by immersing the reactive surface or the
measuring sensor (see example 4) into the reaction mixture. The
temperature changes in the reactive surface are programmed such
that 50 heating and cooling cycles can be carried out within ca. 1
minute. The surface is freed of all non-covalently bound reaction
partners by subsequent immersion of the measuring sensor into a
solution of distilled water and briefly heating it. For analysis
the surface purified in this way that now only contains primer
molecules and extended primer molecules is introduced together with
the measuring sensor into an apparatus which enables a direct
determination of the amount of extended product based on plasmon
resonance.
LIST OF REFERENCE SYMBOLS
1. device, measuring unit according to the invention
2. reaction mixture
3. reaction vessel
4. cooling element
5. heating element/surface
6. oligonucleotides
7. connections for the cooling element
8. connections for the heating element
9. replaceable measuring sensor
10. electronic connection and cooling system
11. holder for reaction vessel
12. stirrer
13. coolant
14. supporting and separating filter (separating the coolant)
__________________________________________________________________________
# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii)
NUMBER OF SEQUENCES: 3 - - - - (2) INFORMATION FOR SEQ ID NO: 1: -
- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 nucleoti - #des (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -
- (ii) MOLECULE TYPE: nucleic acid - - (xi) SEQUENCE DESCRIPTION:
SEQ ID NO: - #1: - - GAAGGGAGGA AGGAGGGAGC GGAC - # - # 24 - - - -
(2) INFORMATION FOR SEQ ID NO: 2: - - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 nucleoti - #des (B) TYPE: nucleic acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE:
nucleic acid - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2: - -
GAAGGGAGGA AGGAGGGAGC GGACGTCCAC CACCACCCAA - # CCACCCCACC C 51 - -
- - (2) INFORMATION FOR SEQ ID NO: 3: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 nucleoti - #des (B) TYPE: nucleic
acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii)
MOLECULE TYPE: nucleic acid - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO: - #3: - - GGGTGGGGTG GTTGGGTGGT GGTG - # - # 24
__________________________________________________________________________
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